JP7233667B2 - Method for producing disintegrating unicellular red algae and medium for disintegrating unicellular red algae - Google Patents

Description

The present invention relates to a method for producing disintegrating unicellular red algae and a medium for disintegrating unicellular red algae.
This application claims priority based on Japanese Patent Application No. 2020-141202 filed in Japan on August 24, 2020, the content of which is incorporated herein.

Microalgae have a high carbon dioxide fixing capacity compared to land plants and do not compete with agricultural products for growing places. etc., are used industrially.
When microalgae are used industrially, it is desirable to use microalgae that can be cultivated outdoors in large quantities from the viewpoint of cost and the like. However, in order for microalgae to be mass-cultivable outdoors, they must be resistant to environmental changes (light, temperature, etc.), can be cultured under conditions where other organisms cannot survive, and can grow to a high density. and other conditions are required.

Some unicellular red algae grow preferentially in sulfuric acid hot springs. Such unicellular red algae are characterized in that they can be cultured in environments such as high salt concentration, high temperature, and low pH where other organisms cannot grow. Therefore, such unicellular red algae are considered suitable for industrial use.

In general, in the case of microalgae having cell walls, it is necessary to destroy the cell walls when extracting intracellular components. Methods for destroying cell walls include physical treatment, chemical treatment, enzymatic treatment, and the like. Therefore, if it is possible to generate cells with almost no cell walls, it is thought that the extraction of intracellular components will become easier. For example, Patent Document 1 describes that disintegrating cells without a strong cell wall could be produced from non-disintegrating cells with a strong cell wall in algae of the class Idycogome, which is a unicellular red alga. there is

WO2019/107385

According to the method described in Patent Document 1, it is difficult to stably maintain disintegrating cells produced from non-disintegrating cells for a long period of time. Therefore, there is a demand for a technology that can stably maintain disintegrating cells for a long period of time.

Accordingly, an object of the present invention is to provide a method for producing disintegrating unicellular red algae and a culture medium for disintegrating unicellular red algae that can stably maintain disintegrating cells.

The present invention includes the following aspects.
[1] A method for producing disintegrating unicellular red algae, which comprises culturing unicellular red algal cells in a medium containing 80 mM or more of an osmotic pressure regulator.
[2] A method for producing disintegrating unicellular red algae, which comprises culturing unicellular red algal cells in a medium having an osmotic pressure of 150 mOsm/kg or more.
[3] The method for producing a disintegrating unicellular red algae according to [1] or [2], wherein the osmotic pressure regulator is at least one selected from the group consisting of sugars, sugar alcohols and amino acids.
[4] The method for producing disintegrating unicellular red algae according to any one of [1] to [3], wherein the unicellular red algal cells are non-disintegrating cells.
[5] The method for producing disintegrating unicellular red algae according to [4], wherein the unicellular red algae cells are polyploid cells.
[6] The method for producing a disintegrating unicellular red algal cell according to any one of [1] to [3], wherein the unicellular red algal cell is a disintegrating cell.
[7] The method for producing a disintegrating unicellular red algal cell according to [6], which is a method for maintaining disintegrating unicellular red algal cells as disintegrating cells.
[8] The method for producing disintegrating unicellular red algae according to [6], which is a method for growing disintegrating unicellular red algal cells.
[9] A medium for disintegrating unicellular red algae containing 80 mM or more of an osmotic pressure regulator.
[10] A medium for disintegrating unicellular red algae, which has an osmotic pressure of 150 mOsm/kg or more.
[11] The medium for disintegrating unicellular red algae according to [9] or [10], wherein the osmotic pressure regulator is at least one selected from the group consisting of sugars, sugar alcohols and amino acids.
[12] The medium for disintegrating unicellular red algae according to any one of [9] to [11], which is used for producing disintegrating unicellular red algal cells from non-disintegrating unicellular red algae cells .
[13] The medium for disintegrating unicellular red algae according to [12], wherein the non-disintegrating unicellular red algal cells are polyploid unicellular red algae cells.
[14] The medium for disintegrating unicellular red algae according to any one of [9] to [11], which is used for maintaining disintegrating unicellular red algal cells as disintegrating cells.
[15] The medium for disintegrating unicellular red algae according to any one of [9] to [11], which is used for growing disintegrating unicellular red algal cells.

INDUSTRIAL APPLICABILITY According to the present invention, a method for producing disintegrating unicellular red algae and a culture medium for disintegrating unicellular red algae capable of stably maintaining disintegrating cells are provided.

Examples of disintegrating cell colonies generated from non-disintegrating unicellular red algal cells are shown. A photograph of an agar plate in which disintegrating cells of the CCCryo127-00 strain were inoculated and subcultured on an 18% sorbitol + Gross 1.5% agar medium is shown. A photograph of a plate in which disintegrating cells of the CCCryo127-00 strain were cultured for one month on an 18% sorbitol + Gross 1.5% agar medium is shown. Fig. 2 shows a photograph of a plate in which disintegrating cells of the CCCryo127-00 strain were cultured for 2 weeks on a 1% sorbitol + Gross 1.5% agar medium. Reversion to non-disintegrating cells was confirmed. An example of growth of disintegrating cells of strain CCCryo127-00 on 18% sorbitol + Gross 1.5% agar medium is shown. The photo on the left is the plate at the start of the culture, and the photo on the right is the plate after 3 weeks of culture. An example of generating disintegrating cells from the non-disintegrating CCCryo127-00 strain on 18% sorbitol + Gross 1.5% agar medium is shown. Disintegrating cells were maintained on subcultured agar. An example of growing disintegrating cells of strain CCCryo127-00 in 18% sorbitol+Gross liquid medium is shown.

The cells described herein can be isolated. "Isolated" means separated from the natural state.

<Method for producing disintegrating unicellular red algae>
A first aspect of the present invention is a method for producing disintegrating unicellular red algae, comprising culturing unicellular red algal cells in a medium containing 80 mM or more of an osmotic pressure regulator.
As used herein, the term "disintegrative cell" means a cell that can be easily destroyed even by mild treatment because it does not have a strong cell wall. A "non-disintegrating cell" means a cell that cannot be easily destroyed by mild treatment, for example, because it has a strong cell wall. The mild treatment referred to here includes, for example, neutralization treatment, hypotonic treatment, freeze-thaw treatment, surfactant treatment, and the like.
An object of the present invention is to provide a method for producing disintegrating unicellular red algae capable of stably maintaining disintegrating cells. In the present invention, it can be judged that "disintegrating cells can be stably maintained" when disintegrating cells can be maintained for more than two weeks.

(unicellular red algae)
"Unicellular red algae" refers to algae that belong to the phylum Rhodophyta and are unicellular. Unicellular red algae include the classes Cyanidiophyceae, Stylonematophyceae, Porphyridiophyceae, and Rhodellophyceae. Among these, Cyanidiophyceae is preferable because it is easy to stably maintain disintegrating cells. Cyanidioschyzon genus, Cyanidium genus, and Galdieria genus are known in the Cyanidioschyzon genus. Cyanidium and Garderia exist in nature as non-disintegrating cells. Therefore, among the genus Idyucogome, the genus Cyanidium and the genus Garderia are preferred, and the genus Garderia is more preferred.
As the Garderia genus, for example, G. sulphuraria, G. partita, G. Daedala, G. maxima and the like, but are not limited to these. As the genus Garderia, G. sulphuraria is particularly preferred.
Cyanidium includes, for example, C. caldarium, C. sp. include, but are not limited to, Monte Rotaro and the like.
Algae strains of the class Idyucogome include, for example, those described in FIG. 10 of WO 2019/107385.

In the method of this embodiment, the unicellular red algae cells used at the start of culture may be non-disintegrating cells or disintegrating cells. A non-disintegrating cell may be a polyploid (eg, diploid) cell.
When non-disintegrating cells are cultured as unicellular red algal cells by the method of this embodiment, disintegrating cells may be generated during the culture. By continuing the culture by the method of this embodiment, the disintegrating cells can be maintained as they are without reverting to non-disintegrating cells. Thus, methods of this embodiment encompass methods of producing disintegrating unicellular red algae from non-disintegrating unicellular red algae cells.
When disintegrating cells are cultured as unicellular red algae cells by the method of this embodiment, they are maintained as disintegrating cells without reverting to non-disintegrating cells during culturing. Thus, the methods of this embodiment encompass methods of maintaining disintegrating unicellular red algae cells.
According to the method of this embodiment, the disintegrating unicellular red algae cells proliferate as disintegrating cells during culture. Thus, the methods of this embodiment encompass methods of growing disintegrating unicellular red algae.

(Culture medium)
The medium used in the method of this aspect is a medium containing 80 mM or more of an osmotic pressure regulator.
"Tonicity modifier" refers to a chemical substance capable of modulating osmotic pressure. The osmotic pressure adjusting agent is not particularly limited as long as it is a chemical substance that can adjust the osmotic pressure by adding it to the medium. Examples of osmotic pressure regulators include sugars, sugar alcohols, amino acids, metal salts, urea, proteins, betaine, inositol, polysaccharides and the like. Among these, sugars, sugar alcohols, amino acids, and metal salts are preferred.

Examples of sugars include dihydroxyacetone, glyceraldehyde, erythrulose, erythrose, threose, ribulose, xylulose, ribose, arabinose, xylose, lyxose, deoxyribose, psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose. , gulose, idose, galactose, talose, fucose, fucrose, rhamnose, sedoheptulose and the like monosaccharides (either D or L, or a mixture of D and L); sucrose, lactulose , lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiose , meribiulose, neolactose, galactosucrose, sylabiose, neohesperidose, rutinose, rutinulose, bisianose, xylobiose, primevelose, trehalosamine, maltitol, cellobionic acid, lactosamine, lactosediamine, lactobionic acid, lactitol, hyalobiuronic acid, sucralose Sugar; trisaccharides such as nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, kestose; tetrasaccharides such as nystose, nigerotetraose, stachyose; Oligosaccharides such as sugars, gentio-oligosaccharides, nigerooligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides, mannan-oligosaccharides, xylooligosaccharides, and soybean oligosaccharides, etc., may be mentioned, but not limited thereto. Preferred sugars are monosaccharides or disaccharides.
Monosaccharides include dihydroxyacetone, glyceraldehyde, erythrulose, erythrose, threose, ribulose, xylulose, ribose, arabinose, xylose, lyxose, deoxyribose, psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, Gulose, idose, galactose, talose, fucose, fucrose, rhamnose, sedoheptulose are preferred, dihydroxyacetone, glyceraldehyde, erythrulose, erythrose, ribulose, ribose, arabinose, xylose, deoxyribose, fructose, glucose, mannose, galactose, or sedoheptulose is more preferred, and glucose is even more preferred.
Disaccharides include sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiose, melibiulose, neolactose, galactosucrose, sylabiose, neohesperidose, rutinose, rutinulose, bisianose, xylobiose, primevelose, trehalosamine, maltitol, cellobionic acid, lactosamine, lactosediamine, lactobionic acid, lactitol , hyalobiuronic acid, or sucralose, more preferably sucrose, lactulose, lactose, maltose, trehalose, or cellobiose, and even more preferably sucrose.

Examples of sugar alcohols include trivalent sugar alcohols such as glycerol; tetravalent sugar alcohols such as erythritol, D-threitol and L-threitol; D-arabinitol, L-arabinitol, xylitol, ribitol, adonitol and the like. pentavalent sugar alcohols; hexavalent sugar alcohols such as D-iditol, galactitol, dulcitol, D-glucitol, sorbitol and mannitol; heptavalent sugar alcohols such as boremitol and perseitol; Examples include, but are not limited to, octahydric sugar alcohols such as octitol; 9hydric sugar alcohols such as isomalt, lactitol and maltitol; and mixtures of sugar alcohols such as HSH and reduced starch syrup. The sugar alcohol is preferably a trivalent sugar alcohol, a tetravalent sugar alcohol, a pentavalent sugar alcohol, a hexavalent sugar alcohol, a nonvalent sugar alcohol, or a mixture of sugar alcohols. A hexavalent sugar alcohol is more preferred, and a hexavalent sugar alcohol is even more preferred.
The sugar alcohol is preferably glycerol, erythritol, xylitol, sorbitol, mannitol, isomalt, lactitol, maltitol, HSH, or reduced starch syrup, more preferably mannitol or sorbitol.

Amino acids may be either D- or L-forms, or may be a mixture of D- and L-forms. Amino acids may be α-amino acids, β-amino acids, γ-amino acids, and δ-amino acids. Examples of amino acids include alanine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine, 2-aminoadipic acid, 3-aminoadipic acid, 2-aminobutanoic acid, 2,4-diaminobutanoic acid, 2-aminohexanoic acid, 6-aminohexanoic acid, β-alanine, 2-aminopentanoic acid, 2,3 -diaminopropanoic acid, 2-aminopimelic acid, 2,6-diaminopimelic acid, citrulline, cysteic acid, 4-carboxyglutamic acid, 5-oxoproline, pyroglutamic acid, homocysteine, homoserine, homoserine lactone, 5-hydroxylysine, allohydroxy Lysine, 3-hydroxyproline, 4-hydroxyproline, alloisoleucine, norleucine, norvaline, ornithine, sarcosine, allothreonine, thyroxine and the like. Preferred amino acids are alanine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, arginine, serine, threonine, selenocysteine, valine, tryptophan, or tyrosine. , glycine, proline, or arginine are more preferred.

Examples of metal salts include alkali metals (sodium, potassium, etc.) or alkaline earth metals (magnesium, calcium, etc.) and inorganic acids (hydrochloric acid, sulfuric acid, carbonic acid, sulfurous acid, nitric acid, etc.) or organic acids (lactic acid, succinic acid, etc.). , acetic acid, etc.). The metal salt is preferably a salt of an alkali metal or an alkaline earth metal and an inorganic acid, more preferably potassium chloride or sodium sulfate, and still more preferably potassium chloride.

Among these, the osmotic pressure regulator is preferably at least one selected from the group consisting of sugars, sugar alcohols, and amino acids because it is easy to add to the medium to adjust the osmotic pressure. Suitable sugars include glucose, sucrose. Suitable sugar alcohols include hexahydric sugar alcohols (eg, mannitol, sorbitol). Suitable amino acids include glycine, proline, arginine.
The osmotic pressure regulators may be used singly or in combination of two or more.

The medium is not particularly limited as long as it contains 80 mM or more of the osmotic pressure regulator. The medium can be prepared, for example, by adding an osmotic pressure regulator to a medium known as a medium for unicellular algae so that the concentration becomes 80 mM or more. Examples of the medium for unicellular algae include, but are not limited to, inorganic salt media containing nitrogen sources, phosphorus sources, trace elements (zinc, boron, cobalt, copper, manganese, molybdenum, iron, etc.) and the like. For example, nitrogen sources include ammonium salts, nitrates, nitrites and the like, and phosphorus sources include phosphates and the like. Examples of such media include Gross medium, 2× Allen medium (Allen MB. Arch. Microbiol. 1959 32: 270-277.), M-Allen medium (Minoda A et al. Plant Cell Physiol. 2004 45: 667-71.), MA2 medium (Ohnuma M et al. Plant Cell Physiol. 2008 Jan;49(1):117-20.), modified M-Allen medium and the like, but are not limited thereto.

The unicellular red algae may be cultured autotrophically under light irradiation, or may be cultured heterotrophically in the dark. In the case of heterotrophic culture, a carbon source (glucose or the like) may be added to the inorganic salt medium as described above.

The concentration of the osmotic pressure regulator in the medium is not particularly limited as long as it is 80 mM or higher. By setting the concentration of the osmotic pressure regulator to 80 mM or more, the disintegrating cells can be stably maintained regardless of the type of the osmotic pressure regulator. The concentration of the osmotic pressure regulator is 100 mM or higher, 110 mM or higher, 120 mM or higher, 130 mM or higher, 140 mM or higher, 150 mM or higher, 160 mM or higher, 170 mM or higher, 180 mM or higher, 190 mM or higher, 200 mM or higher, 210 mM or higher, 220 mM or higher, 230 mM or higher, 240 mM or higher, 250 mM or higher, 260 mM or higher, 270 mM or higher, 280 mM or higher, 290 mM or higher, 300 mM or higher, 310 mM or higher, 320 mM or higher, 330 mM or higher, 340 mM or higher, 350 mM or higher, 360 mM or higher, 370 mM or higher, 380 mM or higher, 390 mM or higher, or 400 mM or more. The upper limit concentration of the osmotic pressure regulator is not particularly limited, and may be the limit value at which it can be dissolved in the medium. From the viewpoint of cell growth rate, the upper limit concentration of the osmotic pressure regulator is, for example, 2 M or less, 1.5 M or less, 1.4 M or less, 1.3 M or less, 1.2 M or less, 1.1 M or less, or 1 M You can: The lower limit and upper limit can be combined arbitrarily. Examples of the concentration range of the osmotic pressure regulator in the medium include 80 mM to 2M. The concentration range of the osmotic pressure regulator is, for example, preferably 100 mM to 1.5 M, more preferably 200 mM to 1.4 M, even more preferably 300 mM to 1.3 M, and particularly preferably 400 mM to 1.3 M.
Unless otherwise specified, the concentration of the osmotic pressure adjusting agent in the medium is the concentration before starting the culture. When two or more osmotic pressure regulators are used in combination, the total content of the two or more osmotic pressure regulators should be 80 mM or more. The same applies to the range exemplified as the concentration of the osmotic pressure regulator.

For example, when the osmotic pressure regulator is glucose, the glucose concentration in the medium is, for example, 200 mM to 2M, preferably 250 mM to 1.7M, more preferably 270 mM to 1.5M. Alternatively, the glucose concentration in the medium is preferably 4-40% by mass, more preferably 5-30% by mass, relative to the total mass (100% by mass) of the medium.
For example, when the osmotic pressure regulator is sucrose, the sucrose concentration in the medium is, for example, 80 mM to 1.1M, preferably 80 mM to 800 mM, more preferably 80 mM to 600M. Alternatively, the concentration of sucrose in the medium is preferably 2 to 40% by mass, more preferably 3 to 30% by mass, even more preferably 3 to 20% by mass, relative to the total mass (100% by mass) of the medium.
For example, when the osmotic pressure regulator is glycerol, the glycerol concentration in the medium is, for example, 200 mM to 800 mM, preferably 300 mM to 600 mM. Alternatively, the glycerol concentration in the medium is preferably 3 to 6% by mass with respect to the total mass (100% by mass) of the medium.
For example, when the osmotic pressure regulator is mannitol, the mannitol concentration in the medium is, for example, 180 mM to 1.5 M, preferably 200 mM to 1.2 M, more preferably 250 mM to 1 M. Alternatively, 4 to 20% by mass is preferable, and 5 to 18% by mass is more preferable.
For example, when the osmotic pressure regulator is sorbitol, the sorbitol concentration in the medium is, for example, 200 mM to 2M, preferably 400 mM to 1.5M, more preferably 430 mM to 1.5M. Alternatively, the sorbitol concentration in the medium is preferably 5-40% by mass, more preferably 8-27% by mass, relative to the total mass (100% by mass) of the medium.
For example, when the osmotic pressure regulator is glycine, the concentration of glycine in the medium is, for example, 100 mM to 2M, preferably 120 mM to 1.5M, more preferably 130 mM to 1M. Alternatively, the glycine concentration in the medium is preferably 0.5 to 10% by mass, more preferably 1 to 8% by mass, relative to the total mass (100% by mass) of the medium.
For example, when the osmotic pressure regulator is proline, the proline concentration in the medium is, for example, 80 mM to 2M, preferably 500 mM to 1.5M, more preferably 600 mM to 1.5M. Alternatively, the proline concentration in the medium is preferably 1-20% by mass, more preferably 7-10% by mass, relative to the total mass (100% by mass) of the medium.
For example, when the osmotic pressure regulator is arginine, the concentration of arginine in the medium is, for example, 20 mM to 2M, preferably 30 mM to 1.5M, more preferably 50 mM to 1M. Alternatively, the arginine concentration in the medium is preferably 0.5 to 30% by mass, more preferably 1 to 20% by mass, relative to the total mass (100% by mass) of the medium.
For example, when the osmotic pressure regulator is potassium chloride, the potassium chloride concentration in the medium is, for example, 50 mM to 1.5 M, preferably 100 mM to 1 M, more preferably 130 mM to 500 mM. Alternatively, the potassium chloride concentration in the medium is preferably 0.5 to 10% by mass, more preferably 1 to 5% by mass, relative to the total mass (100% by mass) of the medium.

The medium preferably has an osmotic pressure of 150 mOsm/kg or more. By setting the osmotic pressure of the medium to 150 mOsm/kg or more, the disintegrating cells can be stably maintained regardless of the type of osmotic pressure regulator. The osmotic pressure is 200 mOsm/kg or higher, 210 mOsm/kg or higher, 220 mOsm/kg or higher, 230 mOsm/kg or higher, 240 mOsm/kg or higher, 250 mOsm/kg or higher, 260 mOsm/kg or higher, 270 mOsm/kg or higher, 280 mOsm/kg or higher, 290 mOsm or higher. /kg or more, 300 mOsm/kg or more, 310 mOsm/kg or more, 320 mOsm/kg or more, 330 mOsm/kg or more, 340 mOsm/kg or more, 350 mOsm/kg or more, 360 mOsm/kg or more, 370 mOsm/kg or more, 380 mOsm/kg or more, 390 mOsm /kg or more, or 400 mOsm/kg or more. The upper limit of the osmotic pressure is not particularly limited, and may be the limit at which the osmotic pressure regulator can be dissolved in the medium. From the viewpoint of cell growth rate, the upper limit of the osmotic pressure can be, for example, 2000 mOsm/kg or less, 1500 mOsm/kg or less, or 1400 mOsm/kg or less. The lower limit and upper limit can be combined arbitrarily. The osmotic pressure range of the medium includes, for example, 150 to 2000 mOsm/kg. The range of osmotic pressure is, for example, preferably 200 to 1500 mOsm/kg, more preferably 250 to 1400 mOsm/kg, even more preferably 300 to 1400 mOsm/kg, and particularly preferably 400 to 1400 mOsm/kg.
Unless otherwise specified, the osmotic pressure of the medium is the value before starting the culture. The osmotic pressure of the medium can be measured using an osmometer.

The medium may be a liquid medium or a solid medium. As a solid medium, for example, an agar medium can be used. In the case of a solid medium, the above concentration and osmotic pressure of the osmotic pressure adjusting agent may be those in the liquid medium before adding the solidifying agent (eg, agar).

The media exemplified above can be used for generating disintegrating cells from non-disintegrating cells, maintaining disintegrating cells, and growing disintegrating cells.
When producing disintegrating cells from non-disintegrating cells, for example, the concentration of the osmotic pressure regulator is preferably 50 mM to 2M, more preferably 100 mM to 1.5M. The osmotic pressure of the medium is preferably 150-2610 mOsm/kg, more preferably 300-1700 mOsm/kg. The medium may be a liquid medium or a solid medium, but it is preferable to use a solid medium because it is easy to determine that the disintegrating cells have been generated.
When maintaining disintegrating cells, for example, the concentration of the osmotic pressure regulator is preferably 50 mM to 2M, more preferably 100 mM to 1.5M. The osmotic pressure of the medium is preferably 150-2610 mOsm/kg, more preferably 300-1700 mOsm/kg. The medium may be a liquid medium or a solid medium, but it is preferable to use a solid medium because it is easy to stably maintain for a long period of time.
When proliferating disintegrating cells, for example, the concentration of the osmotic pressure regulator is preferably 50 mM to 2M, more preferably 100 mM to 1.5M. The osmotic pressure of the medium is preferably 150-2610 mOsm/kg, more preferably 300-1700 mOsm/kg. The medium may be a liquid medium or a solid medium, but it is preferable to use a liquid medium because cells grow easily.

(Culture conditions)
The method of this aspect includes the step of culturing unicellular red algal cells in a medium containing 80 mM or more of an osmotic pressure regulator. The culture conditions in the culture are not particularly limited, and conditions commonly used as culture conditions for unicellular red algae can be used. Culture conditions include, for example, pH 1-8, temperature 10-50° C., CO ₂ concentration 0.3-3%, and the like. Light conditions may be dark when cultured heterotrophically. For autotrophic culture, light conditions include, for example, 5 to 2000 μmol/m ² s.
Culture conditions are not limited to those exemplified above, and can be appropriately selected according to the type of unicellular red algae. For example, when the unicellular red algae is Idycogome, pH conditions include pH 1.0 to 6.0, preferably pH 1.0 to 5.0, and more preferably pH 1.0 to 3.0. Temperature conditions include 15 to 50°C, preferably 30 to 50°C, and more preferably 35 to 50°C. The light intensity is 5 to 2000 μmol/m ² s, preferably 5 to 1500 μmol/m ² s. It may be cultured in continuous light, or a light-dark cycle (10L:14D, etc.) may be provided. In addition, when cultured heterotrophically, it can be cultured in the dark.

The culture period is not particularly limited. When the unicellular red algae cells used at the start of culture are non-disintegrating cells, the cells are cultured at least until disintegrating cells are produced. In the method of this aspect, disintegrating cells can be generated in a short period of time by using a medium containing 80 mM or more of an osmotic pressure regulator. When producing disintegrating cells from non-disintegrating cells, the culture period is preferably 5 days or longer, more preferably 10 days or longer, and even more preferably 14 or 15 days or longer. In the method of this aspect, the disintegrating cells generated during culture are stably maintained as disintegrating cells. Therefore, the upper limit of the culture period is not particularly limited.
When the unicellular red algal cells used at the start of culture are disintegrating cells, the culture period is not particularly limited. In the method of this aspect, since the disintegrating cells are stably maintained, the culture may be continued for the period necessary to maintain the disintegrating cells.

During the culture period, the unicellular red algae cells may be passaged as appropriate. According to the method of this aspect, the disintegrating cells can be stably maintained in the same medium for two weeks or longer. Therefore, the passage interval can be two weeks or longer. For example, by subculturing the disintegrating unicellular red algal cells once every one to three months, the disintegrating cells can be more stably maintained. The passage interval is preferably 1 month to 1.5 months. When proliferating disintegrating cells, passages may be performed at shorter intervals in order to increase the efficiency of proliferation. For example, the passage interval for proliferation is preferably 14 to 60 days, more preferably 14 to 42 days.

(Confirmation method for disintegrating cells)
In the method of this embodiment, disintegrating cells can be produced from non-disintegrating unicellular red algal cells, and the disintegrating cells can be stably maintained for two weeks or longer. The method for confirming that the unicellular red algae cells are disintegrating cells is not particularly limited, and for example, the following methods can be used.

Non-disintegrating cells have a rigid cell wall, whereas disintegrating cells do not. Therefore, by observing the morphology of cells, disintegrating cells can be identified. For example, disintegrating cells usually do not have a cell wall when observed with an optical microscope (for example, at a magnification of 600). Therefore, when no cell wall is observed with an optical microscope, the cells can be determined to be disintegrating cells.
Disintegrating cells can be destroyed by relatively mild treatments (neutralization treatment, hypotonic treatment, freeze-thaw treatment, surfactant treatment, etc.). For example, when cells are suspended in a medium containing 2% by mass of a surfactant and the cells disintegrate immediately to 5 minutes after the addition of the surfactant, the cells can be determined to be disintegrating cells. Examples of the surfactant include sodium dodecyl sulfate. More specifically, sodium dodecyl sulfate is added to the culture medium of unicellular red algal cells to a concentration of 2% by mass, and if the cells disintegrate within 5 minutes after addition, the cells are disintegrating cells. can judge. Whether or not the cells have disintegrated can be confirmed by observing the cells with an optical microscope.
In addition, when the cells are cultured on a solid medium, it is possible to determine whether the cells are disintegrating cells by the shape of the colonies. Disintegrating cells usually do not have a rigid cell wall, resulting in a flattened shape that extends over the surface of the solid medium compared to colonies of non-disintegrating cells. If a colony with such a shape appears on the solid medium, it can be determined to be a colony of disintegrating cells.

According to the method of this embodiment, disintegrating unicellular red algae can be produced from non-disintegrating unicellular red algae, and the disintegrating unicellular red algae can be stably maintained. In addition, disintegrating unicellular red algae can be propagated as disintegrating cells.
When disintegrating unicellular red algae are cultured in a normal medium, cells reverting to non-disintegrating cells appear and non-disintegrating cells proliferate. Therefore, it is necessary to select disintegrating cells and repeat subculturing at intervals of about 5 days. On the other hand, according to the method of this aspect, the appearance of cells that revert to non-disintegrating cells is suppressed, and disintegrating cells are retained for more than two weeks (preferably one month or more) without passage. can be maintained.

The disintegrating unicellular red alga produced, maintained or grown by the method of this embodiment can easily destroy cells under mild conditions. Therefore, cell components can be easily extracted. In addition, even if the disintegrating single-celled red algae produced, maintained or grown by the method of this aspect are directly added to foods or functional foods without performing cell wall destruction treatment, the intracellular components are efficiently digested and absorbed. be done.

<Medium for disintegrating unicellular red algae>
A second aspect of the present invention is a medium for disintegrating unicellular red algae containing 80 mM or more of an osmotic pressure regulator.

The medium of this embodiment is the same as that described in the above "<Method for Producing Disintegrating Unicellular Red Algae>". The medium of this embodiment can be used to generate disintegrating unicellular red algae cells from non-disintegrating unicellular red algae cells. Also, disintegrating unicellular red algal cells can be used to maintain disintegrating cells. Also, disintegrating unicellular red algae cells can be used for propagation.

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

<Unicellular red algae>
Galdieria sulphuraria CCCryo127-00 strain (hereinafter also referred to as “CCCryo127-00 strain”) was used as the unicellular red algae.

<Culture medium>
Gross medium was used as the basal medium. Table 1 shows the composition of Gross medium. Tables 2 and 3 show the compositions of the Fe-EDTA Solution and Trace Elements used in the Gross medium, respectively.

<Method for confirming disintegrating cells>
Cells were suspended in Gross medium containing 2% by mass of a surfactant (sodium dodecyl sulfate (SDS)), and cells that disintegrated were determined to be disintegrating cells. Disintegration of cells was confirmed by observation using an optical microscope. Observation by optical microscopy was performed immediately after the addition of SDS. In addition, colonies in which a large number of disintegrating cells occupy a majority are flattened and have a shape that spreads over the surface of the agar medium compared to colonies formed from non-disintegrating cells (see Fig. 1: colonies of disintegrating cells are indicated by arrows). Some non-disintegrating cells remain in the central part). Therefore, the morphology of the colonies on the agar medium was also used to determine whether the cells were disintegrating cells.

(1) Culture of disintegrating unicellular red algae cells In order to examine the possibility that osmotic pressure affects the stable maintenance of disintegrating cells, sorbitol was used as an osmotic pressure regulator to adjust the osmotic pressure of the medium. It was adjusted. Disintegrating cells were harvested from a colony of disintegrating cells of strain CCCryo127-00 and transferred to 18% sorbitol + Gross 1.5% agar medium. After that, the cells were cultured at 40° C. in the dark in an air atmosphere for 1 to 2 months. Colonies of disintegrating cells were maintained during the culture period (Fig. 2). This result confirms that the disintegrating cells can be stably maintained for more than two weeks by increasing the osmotic pressure in the medium.

(2) Examination of media capable of maintaining disintegrating cells The osmotic pressure regulators shown in Table 4 were added to Gross medium or 1% glucose + Gross medium at 1 to 50% by mass. Furthermore, 1.5% by mass of agar was added to each medium to prepare 1.5% agar medium. Disintegrating cells of strain CCCryo127-00 were seeded on these agar media and cultured at 40° C. in the dark in an air atmosphere for one month or more. During the culture period, the morphology of the colonies on the agar medium was observed to confirm whether colonies of disintegrating cells were maintained. We also assessed the proliferation of disintegrating cells based on colony size. The results are shown in Table 4 based on the following evaluation criteria.

<Evaluation Criteria>
A: Disintegrative cells maintained for 1 month or more B: Disintegrated cells maintained for more than 2 weeks and less than 1 month C: Disintegrated cells maintained for 2 weeks or less N: No proliferation D: Cannot be cultured (dead)
-: Not implemented

FIG. 3 shows an example in which colonies of disintegrating cells are maintained. FIG. 3 is a plate cultured on 18% sorbitol+Gross 1.5% agar medium for one month.
An example of regression to colonies of non-disintegrating cells is shown in FIG. FIG. 4 is a plate cultured for 2 weeks on 1% sorbitol+Gross 1.5% agar medium.
FIG. 5 shows an example of growth of colonies of disintegrating cells. Figure 5 is a plate cultured on 18% sorbitol + Gross 1.5% agar. The photo on the left is the plate at the start of the culture, and the photo on the right is the plate after 3 weeks of culture.

In Table 4, "Gross" indicates Gross medium, and "1% Glc+Gross" indicates 1% glucose Gross medium. The numbers in parentheses represent the molar concentration of the osmotic pressure regulator.

Table 5 shows the results of measuring the osmotic pressure of each medium shown in Table 4 before adding agar. The osmotic pressure of the medium was measured with an osmometer (product name: automatic osmotic pressure analyzer Ozmostation OM-6060, manufacturer: ARKRAY, Inc.). In Table 5, the values in brackets [] indicate the osmotic pressure (mOsm/kg).

From the results in Table 4, it was confirmed that the medium to which about 80 mM or more of the osmotic pressure regulator was added was able to maintain disintegrating cells for more than 2 weeks. Higher concentrations of the osmotic agent tended to slow growth, but even when the osmotic agent was added up to the upper solubility limit, we were generally able to maintain disintegrating cells for more than 2 weeks. Considering the growth rate, the upper limit of the concentration of the osmotic pressure regulator was thought to be about 1.5M.

From the results in Table 5, it was confirmed that the medium with an osmotic pressure of about 150 mOsm/kg or more could maintain disintegrating cells for more than two weeks. Higher osmolarity tended to slow growth, but even at higher osmolarity, disintegrating cells could generally be maintained for more than one month. Considering the growth rate, the upper limit of the osmotic pressure of the medium was thought to be about 1500 Osm/kg.

(3) Production of disintegrating unicellular red algal cells using the medium studied in (2) Non-disintegrating cells of strain CCCryo127-00 were seeded on 18% sorbitol + Gross 1.5% agar medium. After that, the cells were cultured at 40° C. in the dark in an air atmosphere. During the culture period, it was confirmed whether colonies of disintegrating cells appeared.
As a result, colonies of disintegrating cells appeared in about two weeks (FIG. 6, colonies of disintegrating cells are indicated by arrows). Cells were picked from a colony of disintegrating cells and plated onto 18% sorbitol + Gross 1.5% agar. It was confirmed that colonies of disintegrating cells could be maintained for more than one month even in the subcultured medium (Fig. 6).

These results indicated that the use of the medium examined in (2) enabled efficient production of disintegrating cells from non-disintegrating cells.

(4) Liquid Culture Using Medium Considered in (2) CCCryo127-00 strain disintegrating cells were seeded in 18% sorbitol+Gross liquid medium. After that, the cells were cultured at 40° C. in the dark in an air atmosphere. As a result, it was confirmed that the cells proliferate satisfactorily while maintaining the disintegrating cells (Fig. 7).

While the preferred embodiments of the invention have been described and illustrated, it is to be understood that they are intended to be illustrative of the invention and should not be taken as limiting. Additions, omissions, substitutions, and other changes can be made without departing from the spirit or scope of the invention. Accordingly, the present invention should not be viewed as limited by the foregoing description, but only by the scope of the appended claims.

Claims (15)

culturing unicellular red algal cells in a medium containing 80 mM or more of an osmotic pressure regulator;
after said culturing, harvesting the disintegrating cells;
At least the osmotic agent is selected from the group consisting of monosaccharides (excluding glucose), disaccharides (excluding sucrose) , sugar alcohols (excluding glycerol) , amino acids, and metal salts (excluding sodium salts). is a kind of
The unicellular red algae are Garderia algae,
When the osmotic pressure adjusting agent is a monosaccharide (excluding glucose) or a disaccharide (excluding sucrose), the concentration of the osmotic pressure adjusting agent in the medium is 1.4 M or less,
When the osmotic pressure adjusting agent is a sugar alcohol (excluding glycerol), the concentration of the osmotic pressure adjusting agent in the medium is 1 M or less,
When the osmotic pressure adjusting agent is an amino acid, the concentration of the osmotic pressure adjusting agent in the medium is 2M or less,
When the osmotic pressure adjusting agent is a metal salt (excluding sodium salt), the concentration of the osmotic pressure adjusting agent in the medium is 2 M or less.
A method for producing disintegrating unicellular red algae. culturing unicellular red algal cells in a medium having an osmotic pressure of 150 to 2610 mOsm/kg or more;
after said culturing, harvesting the disintegrating cells;
The medium contains at least one permeant selected from the group consisting of monosaccharides (excluding glucose), disaccharides (excluding sucrose) , sugar alcohols (excluding glycerol) , amino acids, and metal salts (excluding sodium salts). containing a pressure regulator,
The unicellular red algae are Garderia algae,
A method for producing disintegrating unicellular red algae. Claim 1 or 2, wherein the osmotic pressure regulator is at least one selected from the group consisting of monosaccharides (excluding glucose), disaccharides (excluding sucrose) , sugar alcohols (excluding glycerol) , and amino acids. 3. The method for producing the disintegrating unicellular red algae according to . The method for producing a disintegrating unicellular red algal cell according to any one of claims 1 to 3, wherein the unicellular red algal cell is a non-disintegrating cell. 5. The method for producing a disintegrating unicellular red alga according to claim 4, wherein the non-disintegrating cells are polyploid cells. The method for producing a disintegrating unicellular red algal cell according to any one of claims 1 to 3, wherein the unicellular red algal cell is a disintegrating cell. The method for producing a disintegrating unicellular red algal cell according to claim 6, wherein the disintegrating unicellular red algal cell is maintained as a disintegrating cell. The method for producing a disintegrating unicellular red algal cell according to claim 6, which is a method for growing disintegrating unicellular red algal cells. Containing an osmotic pressure regulator of 80 mM or more,
At least the osmotic agent is selected from the group consisting of monosaccharides (excluding glucose), disaccharides (excluding sucrose) , sugar alcohols (excluding glycerol) , amino acids, and metal salts (excluding sodium salts). A medium for disintegrating unicellular red algae , which is a kind of
The unicellular red algae are Garderia algae,
When the osmotic pressure adjusting agent is a monosaccharide (excluding glucose) or a disaccharide (excluding sucrose), the concentration of the osmotic pressure adjusting agent in the medium is 1.4 M or less,
When the osmotic pressure adjusting agent is a sugar alcohol (excluding glycerol), the concentration of the osmotic pressure adjusting agent in the medium is 1 M or less,
When the osmotic pressure adjusting agent is an amino acid, the concentration of the osmotic pressure adjusting agent in the medium is 2M or less,
When the osmotic pressure adjusting agent is a metal salt (excluding sodium salt), the concentration of the osmotic pressure adjusting agent in the medium is 2 M or less.
Medium for disintegrating unicellular red algae . Osmotic pressure is 150 to 2610 mOsm/kg or more,
At least one osmotic pressure regulator selected from the group consisting of monosaccharides (excluding glucose), disaccharides (excluding sucrose), sugar alcohols (excluding glycerol) , amino acids, and metal salts (excluding sodium salts) including
The unicellular red algae are Garderia algae,
Medium for disintegrating unicellular red algae. 11. According to claim 9 or 10, wherein the osmotic pressure regulator is at least one selected from the group consisting of monosaccharides (excluding glucose), disaccharides (excluding sucrose) , sugar alcohols (excluding glycerol) , and amino acids. A medium for disintegrating unicellular red algae as described. The medium for disintegrating unicellular red algae according to any one of claims 9 to 11, which is used for producing disintegrating unicellular red algal cells from non-disintegrating unicellular red algal cells. 13. The disintegrating unicellular red algal medium of claim 12, wherein the non-disintegrating unicellular red algae cells are polyploid unicellular red algae cells. The medium for disintegrating unicellular red algae according to any one of claims 9 to 11, which is used for maintaining disintegrating unicellular red algal cells as disintegrating cells. The medium for disintegrating unicellular red algae according to any one of claims 9 to 11, which is used for growing disintegrating unicellular red algal cells.

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